Abstract: Objective To determine if levels of endogenous estrogen or estrogen metabolites are associated with an increased risk of developing knee osteoarthritis (OA) in women. Methods Serum estradiol (E 2 ) and 2 urinary estrogen metabolites (2-hydroxyestrone and 16Α-hydroxyestrone) with radiographically defined prevalent and incident knee OA in 842 white and African American women from the Southeast Michigan Arthritis Cohort. Results The mean age and body mass index (BMI) of women in the cohort were 42.3 years and 28.5 kg/m 2 , respectively. Women who developed radiographically defined knee OA had significantly greater odds of having baseline endogenous early follicular phase estradiol concentrations in the lowest tertile (<47 pg/ml; odds ratio [OR] 1.88, 95% confidence interval [95% CI] 1.07–3.51) compared with those with estradiol concentrations in the middle tertile [47–77 pg/ml]), after adjustment for age, BMI, and other covariates. Women who developed knee OA also had greater odds of having baseline urinary concentrations of 2-hydroxyestrone in the lowest tertile (OR 2.9, 95% CI 1.49–5.68) compared with women with 2-hydroxyestrone concentrations in the middle tertile), after adjustment for covariates. Women who developed knee OA were more likely to have a ratio of 16Α-hydroxyestrone to 2-hydroxyestrone in the highest tertile (>0.86; OR 1.86, 95% CI 1.01–3.44 compared with women with ratios in the 0.54–0.86 range), after adjustment for other covariates. Conclusion There were significant associations of lower baseline serum estradiol and urinary 2-hydroxyestrone with developing knee OA in middle-aged women.